The tumor suppressor p53 is a central hub in multiple cancer-related signaling networks that control cell proliferation and cell fate decisions. However, the more we learn about p53 the more complex and dynamic its role becomes. Recent studies have revealed that p53 is involved in an increasingly diverse range of cellular fates, including necrosis and senescence in addition to apoptosis. The remarkable biochemical complexity of p53 is essential to its diverse physiological roles, yet our understanding of these mechanisms is far from complete. For example, as shown in studies by dozens of laboratories, non-nuclear p53 functions are nearly as important as DNA transactivation with regard to cell fate signaling. In fact, p53 can induce apoptosis in cells through transcription-independent mechanisms even without a functional DNA binding domain, in the absence of RNA synthesis, protein translation, or even without a nucleus. However, the mechanistic details of this process remains much less characterized than classical p53 DNA transactivation response and warrants further investigation. Oxidation-reduction (redox) reactions are essential biochemical processes in many pro- and anti-apoptotic cellular programs that direct cancer cell metabolism, tumor growth, angiogenesis and metastasis. The specificity of these reactions is primarily defined by the high oxidizability of reactive sulfhydryls in select proteins. p53 is a zinc finger protein with ten cysteines, all within its DNA binding domain, and redox-regulated is widely speculated to play a role in its regulation. However, due to technological limitations, nearly all of the studies assessing p53 redox regulation and function have investigated isolated, recombinant protein. There is very little evidence that p53 is redox-regulated in cells on one of its cysteines under physiologically-relevant conditions or stresses. Using a novel mass spectrometry-based redox analysis technology, OxMRM, as well as non-reducing SDS-PAGE, we've discovered that p53 is redox-regulated in a cellular context in response to DNA damage and oxidative stress. These results, along with our biochemical and functional studies, suggest that redox- regulation of p53 is a critical step in cell fate signaling including apoptosis, senescence, and necrosis. This proposal will further characterize the redox-regulation of p53, including 1) identification of the disulfide-linked p53 partner, 2) quantify and define the redox-status of p53 at the mitochondria, and 3) determine the stage of transcription-independent p53 signaling in which redox-regulation is essential. In the long term, altered redox homeostasis is a hallmark of carcinogenesis and may impact p53 function. For example, deregulation of p53 redox-regulation is a potential mechanism by which DNA damaged cells could evade apoptosis and drive tumorigenesis.